Method and apparatus for tissue grafting and copying

ABSTRACT

Exemplary embodiments of apparatus and method for obtaining one or more portions of biological tissue (“micrografts”) to form grafts are provided. For example, a hollow tube can be inserted into tissue at a donor site, and a pin provided within the tube can facilitate controlled removal of the micrograft from the tube. Micrografts can be harvested and directly implanted into an overlying biocompatible matrix through coordinated motion of the tube and pin. A needle can be provided around the tube to facilitate a direct implantation of a micrograft into a remote recipient site or matrix. The exemplary apparatus can include a plurality of such tubes and pins for simultaneous harvesting and/or implanting of a plurality of micrografts. The harvested micrografts can have a small dimension, e.g., less than about 1 mm, which can promote healing of the donor site and/or viability of the harvested tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/711,936 filed on May 14, 2015, which is a divisional applicationof U.S. patent application Ser. No. 13/102,711 filed May 6, 2011 whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/332,230 filed May 7, 2010, U.S. Provisional Patent Application Ser.No. 61/373,498 filed Aug. 13, 2010, and U.S. Provisional PatentApplication Ser. No. 61/437,507 filed Jan. 28, 2011, the disclosures ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE DISCLOSURE

The present disclosure relates to exemplary embodiments of method andapparatus for providing tissue grafts and reproducing tissue structuresusing tissue, e.g., from a donor site.

BACKGROUND INFORMATION

An autograft can refer to tissue transplanted from one part of anindividual's body (e.g., a “donor site”) to another part (e.g., a“recipient site”). Autografts can be used, for example, to replacemissing skin and other tissue and/or to accelerate healing resultingfrom trauma, wounds, burns, surgery and birth defects. Availability oftissue for autografting can be limited by characteristics of candidatedonor sites, including a number and/or total area of tissue grafts,healing behavior of the donor site, similarity of the donor andrecipient sites, aesthetic considerations, etc.

Skin grafting can be performed surgically. For example, a conventionalautograft procedure may include excision or surgical removal of burninjured tissue, choosing a donor site, which may be an area from whichhealthy skin is removed to be used as cover for the cleaned burned area,and harvesting, where the graft may be removed from the donor site,e.g., using an instrument similar to an electric shaver. Such instrument(e.g., a dermatome) can be structured to gently shave a piece of tissue,which may be, e.g., about 10/1000 of an inch thick for a split-thicknessgraft, from the skin at the unburned donor site to use as a skin graft.The skin graft can then be placed over the cleaned wound so that it canheal. Donor skin tissue can be removed to such a depth that the donorsite can heal on its own, in a process similar to that of healing of asecond degree burn.

Two conventional types of autografts which may be used for a permanentwound coverage include sheet grafts and meshed grafts. A sheet graft canrefer to a piece of skin tissue removed from an undamaged donor site ofthe body, in a process that may be referred to as harvesting. The sizeof the donor skin piece that is used may be about the same size as thedamaged area. The sheet graft can be applied over the excised wound, andstapled or otherwise fastened in place. The donor skin tissue used insheet grafts may not stretch significantly, and a sheet graft can beobtained that is slightly larger than the damaged area to be coveredbecause there may often be a slight shrinkage of the graft tissue afterharvesting.

Sheet grafts can provide an improved appearance of the repaired tissuesite. For example, sheet grafts may be used on large areas of the face,neck and hands if they are damaged, so that these more visible parts ofthe body can appear less scarred after healing. A sheet graft may beused to cover an entire burned or damaged region of skin, e.g., if thedamaged site is small. Small areas of a sheet graft can be lost afterplacement because of a buildup of fluid (e.g., a hematoma) can occurunder the sheet graft following placement the sheet graft.

Sheet grafts may be full-thickness or split-thickness. For example,split-thickness skin grafts can be used to cover wounds in burn and skinulcer patients. A conventional split-thickness graft can be formed,e.g., by harvesting a sheet of epidermis and upper dermal tissue from adonor site, in a procedure similar to that of peeling an apple. Thesplit-thickness graft can then be placed on the location of the burn orulcer. The skin tissue may then grow back at the donor site following agenerally extended healing time. Split-thickness grafts may bepreferable to full-thickness grafts because removing large amounts offull-thickness skin tissue from the donor site can lead to scarring andextensive healing times at the donor site, as well as an increased riskof infection. However, skin tissue removed from the donor site for asplit-thickness skin autograft can include only a thin epithelial layer,which can lack certain elements of the dermis that improve structuralstability and normal appearance in the recipient site.

Full-thickness skin grafts can be formed using sheets of tissue thatinclude the entire epidermis layer and a dermal component of variablethickness. Because the dermal component can be preserved infull-thickness grafts, more of the characteristics of normal skin can bemaintained following the grafting procedure. Full-thickness grafts cancontain a greater collagen content, dermal vascular plexus, andepithelial appendages as compared to split-thickness grafts. However,full-thickness grafts can require more precise conditions for survivalbecause of the greater amount of tissue requiring revascularization.

Full-thickness skin grafts can be preferable for repairing, e.g.,visible areas of the face that may be inaccessible by local flaps, orfor graft procedures where local flaps are contraindicated. Suchfull-thickness skin grafts can retain more of the characteristics ofnormal skin including, e.g., color, texture, and thickness, as comparedto split-thickness grafts. Full-thickness grafts may also undergo lesscontraction while healing. These properties can be important on morevisible areas such as the face and hands. Additionally, full-thicknessgrafts in children can be more likely to grow with the individual.However, the application of conventional full-thickness skin grafts canbe limited to relatively small, uncontaminated, well-vascularizedwounds, and thus may not be appropriate for as many types of graftprocedures as split-thickness grafts. Additionally, donor sites forfull-thickness grafts can require surgical closure or resurfacing with asplit-thickness graft.

A meshed skin graft can be used to cover larger areas of open woundsthat may be difficult to cover using sheet grafts because of, e.g., alack of a sufficient area of healthy donor sites. Meshing of a skingraft can facilitate skin tissue from a donor site to be expanded tocover a larger area. It also can facilitate draining of blood and bodyfluids from under the skin grafts when they are placed on a wound, whichmay help prevent graft loss. The expansion ratio (e.g., a ratio of theunstretched graft area to the stretched graft area) of a meshed graftmay typically be between about 1:1 to 1:4. For example, donor skin canbe meshed at a ratio of about 1:1 or 1:2 ratio, whereas larger expansionratios may lead to a more fragile graft, scarring of the meshed graft asit heals, and/or extended healing times.

A conventional graft meshing procedure can include running the donorskin tissue through a machine that cuts slits through the tissue, whichcan facilitate the expansion in a pattern similar to that of fishnetting or a chain-link fence. Healing can occur as the spaces betweenthe mesh of the stretched graft, which may be referred to as gaps orinterstices, fill in with new epithelial skin growth. However, meshedgrafts may be less durable graft than sheet grafts, and a large mesh canlead to permanent scarring after the graft heals.

To help the graft heal and become secure, the area of the graft canpreferably be made immobile (e.g., not moved) for at least about fivedays following each surgery. During this immobilization period, bloodvessels can grow from underlying tissue into the skin graft, and canhelp to bond the two tissue layers together. About five days after thegraft is placed, exercise therapy programs, tub baths, and other normaldaily activities can often be resumed. Deep second-degree andfull-thickness burns may require skin graft surgery for quick healingand minimal scarring. Large burn sizes can lead to more than onegrafting procedure during a hospital stay, and may require long periodsof immobilization for healing.

As an alternative to autografting, skin tissue obtained fromrecently-deceased people (which may be referred to, e.g. as a homograft,an allograft, or cadaver skin) can be used as a temporary cover for awound area that has been cleaned. Unmeshed cadaver skin can be put overthe excised wound and stapled in place. Post-operatively, the cadaverskin may be covered with a dressing. Wound coverage using cadavericallograft can then be removed prior to permanent autografting.

A xenograft or heterograft can refer to skin taken from one of a varietyof animals, for example, a pig. Heterograft skin tissue can also be usedfor temporary coverage of an excised wound prior to placement of a morepermanent autograft, and may be used because of a limited availabilityand/or high expense of human skin tissue. In some cases religious,financial, or cultural objections to the use of human cadaver skin mayalso be factors leading to use of a heterograft. Wound coverage using axenograft or an allograft is generally a temporary procedure which maybe used until harvesting and placement of an autograft is feasible.

Epithelial appendages can be regenerated following a grafting procedure.For example, hair can be more likely to grow from full-thickness graftsthan from split-thickness grafts, but such hair growth may beundesirable based on the location of the wound. Accordingly, donor sitesfor full-thickness grafts can be carefully selected based in part, e.g.,on patterns of hair growth at the time of surgery. Further, certain hairfollicles may not be oriented perpendicular to the skin surface, andthey can be transected if an incision provided to remove graft tissue isnot oriented properly.

Sweat glands and sebaceous glands located in graft tissue may initiallydegenerate following grafting. These structures can be more likely toregenerate in full-thickness grafts than in split-thickness graftsbecause full-thickness grafts can be transferred as entire functionalunits. For example, sweat gland regeneration can depend in part onreinnervation of the skin graft with recipient bed sympathetic nervefibers. Once such ingrowth has occurred, the skin graft can assume thesweating characteristics of the recipient site, rather than retainingthe characteristics of the donor site. In contrast, sebaceous glandregeneration may be independent of graft reinnervation and can retainthe characteristics of the donor site. Prior to the regeneration, theskin graft tissue may lack normal lubrication of sebum produced by theseglands, which can make such grafts more susceptible to injury.

In general, grafting procedures may be limited by the amount of tissuewhich can be removed from the donor site without causing excessiveadverse effects. Full-thickness grafts can provide improved tissuequality at the wound site, but the donor site may be more severelydisfigured as described above. Split-thickness grafts can be acompromise between healing times and aesthetic and functional propertiesof the donor and recipient sites, whereas meshing can provide moreextensive graft coverage at the expense of visible scarring.

Harvesting of the graft tissue from the donor site can generallygenerate undesirable large-scale tissue damage to the donor site. On theother hand, small areas of skin wounding adjacent to healthy tissue canbe well-tolerated, and may heal quickly. Such healing of small woundscan occur in techniques such as “fractional photothermolysis” or“fractional resurfacing,” in which patterns of damage having a smalldimension can be created in skin tissue. These exemplary techniques aredescribed, e.g., in U.S. Pat. No. 6,997,923 and U.S. Patent PublicationNo. 2006/0155266. Small-scale damage patterns can heal quickly byregrowth of healthy tissue, and can further provide desirable effectssuch as skin tightening without visible scarring.

In view of the shortcomings of the above described procedures for tissuegrafting, it may be desirable to provide exemplary embodiments of methodand apparatus that can provide tissue suitable for grafting, e.g., whileminimizing unwanted damage to the donor sites.

SUMMARY OF EXEMPLARY EMBODIMENTS

Exemplary embodiments of the present disclosure provide method andapparatus for obtaining small portions of graft tissue that can beaccompanied by rapid healing of the donor site. For example, theexemplary embodiment of the method can be provided for obtaining skingraft tissue by harvesting small portions of the tissue, e.g.,micrografts, from a donor site. Such micrografts can be used to formgrafts or “copy” tissue to generate larger tissue structures from thesmall tissue samples.

Such micrografts can comprise skin tissue that can include, e.g.,epidermal and dermal tissue, and/or tissue obtained from other bodyorgans. The micrografts can have at least one dimension that isrelatively small, e.g., less than about 1 mm, or less than about 0.5 mm,or optionally about 0.3 mm or less, or about 0.2 mm. Such exemplarysmall dimensions of the micrografts can facilitate both healing of thedonor site following harvesting and viability of the micrografts byallowing greater diffusional nourishment of the micrograft tissue. Thesmall regions of damage in the donor site caused by a removal of thetissue portions can heal rapidly with little or no formation of visiblescars. The micrografts obtained from skin tissue can include, e.g.,epidermal and dermal tissue, and can also include stem cells that can belocated proximal to the dermal/fatty layer boundary. The micrografts canalso be obtained from other types of tissue, e.g., various internalorgans or the like.

A fraction of dermal tissue that is removed from a donor site can be,e.g., less than about 70%, or less than about 50%, although otherfractions may be used. The harvested tissue portions can be in the shapeof cylinders, elongated strips, or other geometries which can include atleast one small dimension. In certain exemplary embodiments, a portionof the tissue at the donor site may be frozen or partially frozen. Suchfreezing may facilitate cutting, removal and/or viability of theharvested tissue portions.

In a further exemplary embodiment of the present disclosure, anapparatus for harvesting micrografts can be provided that includes atleast one hollow tube with a pin provided at least partially within thetube, wherein the pin is controllably translatable in a direction alongthe longitudinal axis of the tube. The diameter of the pin can besubstantially the same as the internal diameter of the lumen of thetube, or it can be slightly smaller.

Such tube(s) and corresponding pin(s) can be mechanically coupled to asubstrate having at least one hole therethrough. A plurality of linearactuators can be provided to controllably position and/or move the atleast one tube and pin relative to the substrate. For example, theactuators can be configured to position the at least one tube through ahole in the substrate and control a distance that the distal end of thetube protrudes from a lower surface of the substrate. The actuators canfurther be configured to independently control the position of the pinwithin the tube. The substrate can be configured to be positioned on thesurface of a tissue to facilitate harvesting of micrografts from thetissue and/or implantation of micrografts into the tissue.

In further exemplary embodiments of the present disclosure, a pluralityof tubes and corresponding pins can be provided to facilitate harvestingof a plurality of micrografts. A plurality of actuators or mechanicalarrangements can be provided in communication with the proximal ends ofthe tubes and/or pins to facilitate precise positioning and translationof the plurality of tubes and pins relative to each other and to thetissue. The actuators can be configured to translate all of the tubesand/or pins simultaneously, or optionally to translate certain ones ofthe tubes or pins independently of the other tubes. A vibratingarrangement can be coupled to the apparatus to facilitate the insertionof the tubes into the donor and/or recipient site.

The exemplary micrografts can be placed in a biocompatible matrix, e.g.,to form a graft or larger copy of the donor tissue, or may be implanteddirectly into tissue at the recipient site. The biocompatible matrix canbe formed using collagen, polylactic acid, hyaluronic acid, and/or othersubstances which can support the harvested micrograft tissue portionsand promote their growth. The matrix can optionally include, e.g.,nutrients, growth factors, and/or other substances to promote tissuegrowth or viability. The harvested tissue portions can be bonded to thematrix using techniques such as photochemical tissue bonding to providestructural stability. The matrix can then be applied to the recipientsite, which can promote growth and revascularization of the tissueportions to form a continuous sheet of the grafted tissue. Optionally,the matrix can be placed in a suitable environment to facilitate growthof the micrografts therein, which can then form a larger portion oftissue ‘copied’ from the donor site.

The exemplary micrografts can also be gathered in a compactconfiguration to form graft tissue that can be applied directly to arecipient site. The exemplary micrografts can also be inserted directlyinto the tissue at a recipient site such as, e.g., scar tissue, using,e.g., the exemplary hollow tubes described herein. In certain exemplaryembodiments, the micrografts can be harvested from a donor site tissuethat is different from the tissue type at the recipient site to formvarious types of heterografts.

Still further exemplary embodiments of the present disclosure canprovide methods for extracting or harvesting micrografts from a tissueand optionally place them directly in a matrix material. For example,such exemplary methods can utilize an exemplary apparatus that includesat least one tube and pin provided therein to harvest tissue as follows:

-   -   A) A portion or sheet of a matrix material can be placed on a        donor tissue site, and the distal ends of the tube and pin        provided within the tube can each be positioned proximal to the        upper surface of the matrix.    -   B) The tube and pin can be translated together to penetrate        through the matrix and be positioned proximal to the surface of        the donor site.    -   C) The tube can then be translated downward into the tissue of        the donor site to sever a portion of the tissue from the        surrounding tissue, while the distal end of the pin remains at        the surface of the donor site.    -   D) The tube and pin can then be retracted simultaneously until        the distal end of the tube is proximal to the lower surface of        the matrix material or within the matrix material, and a        micrograft comprising the severed tissue remains within the        distal portion of the tube.    -   E) The tube can then be further retracted from the matrix while        the pin location is held substantially stationary relative to        the matrix, such that the micrograft is held within the matrix        material by the stationary pin and remains within the matrix as        the tube is retracted from around the micrograft.

In yet another exemplary embodiment of the present disclosure, apiercing needle can be provided around at least a portion of the hollowtube and pin. The apparatus including at least one hollow tube, pin, andpiercing needle can be configured to place a micrograft contained withina distal portion of the hollow tube into tissue located at a recipientsite. The piercing needle can be inserted into tissue at a recipientsite. The hollow tube containing the pin and a harvested micrograft inthe distal portion thereof can be advanced through the piercing tubesuch that the distal end of the hollow tube is proximal to or below thesurface of the recipient site. The piercing needle can be withdrawn fromthe recipient site, while the hollow tube is held stationary. The hollowtube can then be withdrawn while holding the pin within the tubestationary relative to the recipient site. In this manner, themicrograft can remain within the tissue of the recipient site, e.g.,held there by the pin as the hollow tube is withdrawn. The pin cansubsequently be withdrawn to leave the micrograft within the recipientsite where the tissue was initially separated by the piercing needle. Asubstrate can be provided with at least one opening therethrough andconfigured to be placed on the tissue surface, to facilitate positioningof the needle, tube and/or pin relative to the tissue being treatedand/or to facilitate mechanical stabilization of the tissue duringtreatment.

These and other objects, features and advantages of the presentdisclosure will become apparent upon reading the following detaileddescription of exemplary embodiments of the present disclosure, whentaken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects, features and advantages of the present disclosure willbecome apparent from the following detailed description taken inconjunction with the accompanying figures showing illustrativeembodiments, results and/or features of the exemplary embodiments of thepresent disclosure, in which:

FIG. 1A is a schematic illustration of an exemplary donor site aftercylindrical portions of micrograft tissue have been harvested therefrom;

FIG. 1B is a schematic illustration of the exemplary donor site shown inFIG. 1A after healing has occurred;

FIG. 1C is a schematic illustration of an exemplary micrograft that canbe removed from the exemplary donor site shown in FIG. 1A;

FIG. 2A is a cross-sectional view of an exemplary graft prepared byproviding harvested micrograft tissue portions in a biocompatiblematrix;

FIG. 2B is a cross-sectional view of the exemplary graft shown in FIG.2A after it has been placed over a wound and some regrowth has occurred;

FIG. 3A is a schematic illustration of another exemplary donor siteafter elongated strips of tissue have been harvested therefrom;

FIG. 3B is a schematic illustration of the exemplary donor site shown inFIG. 3A after healing has occurred;

FIG. 3C is a schematic illustration of an exemplary tissue strip thatmay be removed from the donor site shown in FIG. 3A;

FIG. 4A is a schematic view in plan of a plurality of exemplarycylindrical micrograft tissue portions provided in a compact arrangementto form a graft;

FIG. 4B is a side view of the exemplary micrograft tissue portions shownin FIG. 4A;

FIG. 5A is a schematic illustration of an exemplary apparatus that canbe used to harvest micrograft tissue in accordance with furtherexemplary embodiments of the present disclosure;

FIG. 5B is a schematic illustration of the exemplary apparatus that canbe used to harvest the micrograft tissue in accordance with furtherexemplary embodiments of the present disclosure;

FIG. 6A is a schematic illustration of the exemplary apparatus shown inFIG. 5A that is inserted into an exemplary donor site to harvest anexemplary micrograft;

FIG. 6B is a schematic illustration of the exemplary apparatus shown inFIG. 5A that contains the harvested micrograft;

FIG. 6C is a schematic illustration of the exemplary apparatus shown inFIG. 5A showing the harvested micrograft being removed therefrom;

FIG. 7 is a schematic illustration of the exemplary apparatus that canbe used to harvest micrograft tissue in accordance with furtherexemplary embodiments of the present disclosure;

FIG. 8A is an exemplary image of a distal end of the exemplary apparatusthat includes two points;

FIG. 8B is a further exemplary image of the distal end of the exemplaryapparatus shown in FIG. 8A;

FIGS. 8C and 8D are exemplary schematic illustrations of exemplarygeometric parameters that can be associated with the shape of the distalend of the exemplary apparatus shown in FIG. 8A;

FIGS. 8E-8J are exemplary schematic illustrations of various exemplarygeometric configurations that can be provided for the distal end of theexemplary apparatus shown in FIG. 8A;

FIG. 9 is an exemplary image of the micrografts obtained using theexemplary apparatus shown in FIG. 8A;

FIG. 10A is a schematic illustration of a portion of an exemplaryapparatus that can be used to harvest a micrograft tissue in accordancewith further exemplary embodiments of the present disclosure;

FIG. 10B is an illustration of the portion of the exemplary apparatusshown in FIG. 10A;

FIG. 11 is a schematic illustration of an exemplary apparatus that canbe used to harvest the micrograft tissue in accordance with stillfurther exemplary embodiments of the present disclosure;

FIG. 12 is a schematic illustration of an exemplary sequence ofprocedures that can be used to the harvest micrograft tissue inaccordance with further exemplary embodiments of the present disclosure;

FIG. 13A is a schematic illustration of an exemplary configuration of astabilizing substrate that can be used to harvest the micrograft tissuein accordance with further exemplary embodiments of the presentdisclosure;

FIG. 13B is an exemplary image of a further exemplary configuration of astabilization substrate in accordance with yet further exemplaryembodiments of the present disclosure;

FIG. 14 is a schematic illustration of an exemplary configuration of anarrangement that includes two stabilizing substrates that can be used toharvest the micrograft tissue in accordance with additional exemplaryembodiments of the present disclosure; and

FIGS. 15A-15E are schematic illustrations of an exemplary apparatus andprocedure for placing harvested micrographs directly into tissue at arecipient site;

FIG. 16 is a series of exemplary images of a micrograft harvested frompig skin placed in a collagen gel matrix using the exemplary apparatusand/or method according to exemplary embodiments of the presentdisclosure;

FIG. 17 is a series of exemplary images of micrografts harvested from ablack mouse and implanted in a wound formed in a nude mouse using theexemplary apparatus and/or method according to exemplary embodiments ofthe present disclosure;

FIG. 18A is an exemplary image of a full-thickness skin wound formed ina swine subject;

FIG. 18B is a series of exemplary images showing healing progress of anexemplary wound as shown in FIG. 18A with and without implantedmicrografts;

FIG. 19A is a series of illustrations showing an exemplary sequence ofinsertion and extraction of an exemplary apparatus from a biologicaltissue using the exemplary apparatus and/or method according toexemplary embodiments of the present disclosure; and

FIG. 19B is a plot of exemplary displacement and force data as afunction of time corresponding to the exemplary insertion and extractionsequence shown in FIG. 19A.

Throughout the drawings, the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components, or portions of the illustrated embodiments. Moreover, whilethe present disclosure will now be described in detail with reference tothe figures, it is done so in connection with the illustrativeembodiments and is not limited by the particular embodiments illustratedin the figures.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

According to exemplary embodiments of the present disclosure, methodsand apparatus can be provided for producing autografts, and particularlysuch methods and apparatus which can facilitate more rapid healing ofthe donor site while providing improved tissue characteristics at therecipient site. Exemplary embodiments of the present disclosure caninclude a plurality of small-scale tissue portions (e.g., micrografts)that can be used to provide autografts. Such micrografts can avoidsignificant permanent damage to the donor site while providing grafttissue that can heal rapidly and generate skin tissue having desirableproperties at the recipient site.

In exemplary embodiments of the present disclosure, a method can beprovided for creating autografts in which tissue portions having atleast one small dimension (e.g., micrografts) are harvested from anexemplary donor site 100, as shown in FIG. 1A. The holes 110 shown inFIG. 1A represent regions of the exemplary donor site 100 from whichtissue portions (e.g., micrografts) have been removed. These exemplaryholes 110 can have an approximately round cross-sectional shape,although other shapes may be used.

The exemplary donor site 100 is shown in FIG. 1B after healing of theharvested tissue has occurred. The small regions of damage 100 createdat the donor site by the removed tissue can heal rapidly and/or withoutvisible scarring. For example, the residual pattern of the healed donorsite 100 shown in FIG. 1B may not be easily perceptible by the naked eyeunder normal viewing conditions.

An exemplary micrograft 120 that can be formed, e.g., by harvesting orremoving a portion of the tissue from the donor site 100 to form thehole 110 therein, is shown in FIG. 1C. The exemplary micrograft 120 canhave an elongated shape that may be approximately cylindrical. Themicrografts 120 can include both epidermal tissue 130 and dermal tissue140 from the exemplary donor site 100. For example, the exemplarymicrograft 120 can be about 3 mm in length, which can correspond to atypical total depth of the skin layer (e.g., epidermal and dermallayers). A different length may be used based on the particular skin ortissue characteristics of the donor site 100. In general, it can bepreferable to avoid harvesting a significant amount of subcutaneoustissue, so the harvested micrografts 200 can include primarily theepidermal tissue 130 and the dermal tissue 140. A lower portion 150 ofthe exemplary micrograft 120 can also include stem cells that can bepresent in a lower portion of the dermal layer of the donor site 100(e.g., near a dermal/fatty layer boundary).

A width or diameter of the holes 110 produced during harvesting (whichcan correspond approximately to the diameters of the portions of theharvested micrografts 120) can be less than about 2 mm, or less thanabout 1 mm. In certain exemplary embodiments of the present disclosure,the diameter or width of a micrograft 120 can be less than about 0.5 mm,less than about 0.3 mm. or about 0.2 mm. The size of the exemplary holes110 can be selected, e.g., based on the effects of creating small damageregions in the donor site 100 that can heal rapidly and/or withoutscarring, and on creating portions of tissue that can be small enough topromote viability when transplanted or placed in a growth medium, andlarge enough to form a sufficient amount of graft tissue and/or capturetissue structures that may be present in the donor tissue.

For example, living tissue can be provided with nutrients via adiffusional transport over distances of about 0.1 mm. Accordingly, theexemplary micrografts 120 having at least one dimension that is lessthan about 0.5 mm, e.g., less than about 0.3 mm or, e.g., as small asabout 0.2 mm, can exhibit improved viability and likelihood to survive,and they may grow when used in a graft. Such exemplary micrografts 120can be better able to receive nutrients (including, e.g., oxygen) whenplaced in a recipient site, prior to revascularization of the tissue.

Larger micrografts 120, e.g., those having a width of about 1-2 mm, canalso benefit from such diffusional transport of nutrients, and can alsobe more likely to survive than significantly larger portions of grafttissue (e.g., conventional full-thickness, split-thickness or meshedgrafts). These larger sizes can be preferable for harvested tissue thatis heterogeneous, e.g., tissues that may contain certain structures thatcan be preserved within a single micrograft 120. For example, skintissue has certain structures such as hair follicles, sebaceous glands,etc., and harvesting somewhat larger micrografts 120 from skin may helpto preserve these tissue structures when harvested and transplanted. Onthe other hand, smaller micrografts, e.g. those less than about 0.5 mm,or about 0.2 mm wide, can be suitable for relatively homogeneoustissues, such as muscle tissue, where there are few or no largerstructures in the tissue to be preserved.

A fraction of surface tissue removed from the donor site 100 byharvesting (which can correspond to a fractional surface area of theexemplary donor site 100 occupied by the holes 110) can be less thanabout 70%, or more preferably less than about 50%. The fraction oftissue removed can be sufficiently large to provide enough harvestedmicrografts 120 to form a graft therefrom of appropriate size, but smallenough to facilitate rapid healing at the donor site 100 based on growthfrom the remaining undamaged tissue. Other fractions of tissue can beremoved from a donor site 100 depending on factors such as e.g., theparticular characteristics of the donor site 100, the size of the graftneeded, and the overall amount of donor site tissue available.

In further exemplary embodiments of the present disclosure, a graft 200can be provided by embedding or inserting a plurality of micrografts 120in a biocompatible matrix 210 as shown, e.g., in FIG. 2A. The exemplarymatrix 210 containing the micrografts 120 can be exposed to nutrients topromote growth of the harvested micrografts 120, e.g., to form acontinuous or nearly continuous layer of tissue in the graft 200 aftergrowth has occurred. The exemplary graft 200, which can include thematrix 210 and the micrografts 120, may be placed directly over arecipient site 220 (e.g., a cleaned wound area) as shown in FIG. 2B. Theexemplary micrografts 120 can also include stem cells as describedherein, which can also facilitate healing and integration of theexemplary micrografts 120 when they are transplanted to the recipientsite 220. The recipient site 220 can provide nutrients and/or promoterevascularization of the harvested micrografts 120, which can furtherenhance their growth through the matrix 210 to eventually fill in thespaces separating them. For example, FIG. 2B shows the micrografts 120after they have begun to grow into the surrounding matrix 210.

In one exemplary embodiment of the present disclosure, the micrografts120 can be placed in the matrix 210 at approximately the same spacing(e.g., a similar areal density) as they were removed from the donor site100. This exemplary configuration can generate an amount of graft tissuethat may be approximately the same size as the overall harvested area ofthe donor site 100 after the micrografts 120 grow and fill in the spacesbetween them with new tissue. The average spacing of the micrografts 120in the matrix 210 can also be increased to form a graft tissue that islarger than the overall area of the harvested donor site 100. Theparticular spacing of the micrografts 120 in a particular graft 200 canbe selected based on factors such as, e.g., the size and fractionaldamage of the donor site 100, the size of the recipient site 220 to becovered by the skin graft 200, the time needed for the micrografts 120to regrow and form a continuous tissue layer, the desired appearance ofthe grafted recipient site, etc. For example, the exemplary micrografts120 can be spaced far apart in a particular graft, which can provide alarger graft area but can also require longer healing time and thepossibility of some visible scarring or texture in the healed graft 200.

In a further exemplary embodiment of the present disclosure, tissueportions 320, such as those shown in FIG. 3C, can be harvested in anelongated, narrow strip-like shape. One or more of the exemplary tissuestrips 320 can include both epidermal tissue 130 as well as dermaltissue 140, which can be similar to the micrograft 120 shown in FIG. 1C.For example, the height of the exemplary tissue strip 320 can be about 3mm, or another length that may correspond to a local depth of the dermallayer at the donor site 100. Larger and/or smaller depths of the dermallayer can also be selected when harvesting tissue strips 320 based on,e.g., characteristics of the donor and recipient sites, the wound to berepaired by grafting, etc.

Harvesting of such exemplary tissue strips 320 can leave long, narrowgrooves 310 in a donor region 100 as shown, e.g., in FIG. 3A. A width ofthe grooves 310 (and thus a width of the harvested tissue strips 320)can be less than about 1 mm, or less than about 0.5 mm. In certainexemplary embodiments, the width of such tissue strips can be less thanabout 0.3 mm, or about 0.2 mm. As described herein, such a smalldimension can facilitate diffusional transport of nutrients to the grafttissue and can improve viability of the harvested tissue. A depth of thegrooves 310 from the skin surface can correspond to the height of theharvested strips 320.

A surface area fraction of the exemplary donor site 310 that is removedto form tissue strips 320 can be less than about 70%, or about 50% orless. Factors governing a selection of parameters associated with theharvested elongated tissue strips 320 (e.g., widths and area fractionsremoved from the donor site) may be similar to those described abovewith respect to the substantially cylindrical micrografts 120. Thelength of the harvested strips 320 can be selected based on factors suchas, for example, ease of cutting, removing, and handling the thin tissuestrips 320, the size of the donor site 100, etc. The elongated grooves310 formed in the donor site may also be able to heal rapidly withlittle or no visible scarring, as shown in FIG. 3B, because of the smalllateral dimension and presence of adjacent healthy tissue that cansupport local tissue regrowth.

The harvested strips 320 can be placed, e.g., in a biocompatible matrixsimilar to the matrix 210 shown in FIG. 2A. The tissue strips 320 can bearranged in an approximately parallel configuration, e.g., correspondingto the configuration of the donor-site grooves 310 from which they wereremoved. The spacing between the strips 320 can alternatively beincreased or decreased relative to the spacing of the grooves 310 in thedonor site 100 as desired, e.g., to provide either larger overall areasof graft tissue or more densely packed graft tissue, respectively. Suchharvested tissue strips 320 can be used for certain grafting proceduresbecause the long dimension can preserve structures in the harvested skintissue that may promote revascularization and improve healing of thegraft formed therefrom.

Harvested tissue portions can be removed from the donor site in othershapes, including tile patterns or fractal-like shapes. In general, eachremoved piece of tissue (and, e.g., each corresponding hole or void inthe donor site) can have at least one small dimension that is less thanabout 1 mm, or less than 0.5 mm. In certain exemplary embodiments, thissmall dimension can be less than about 0.3 mm, or about 0.2 mm.

In further exemplary embodiments of the present disclosure, theharvested tissue portions can be placed at the recipient site in a denseconfiguration. For example, FIG. 4A shows a schematic top view of aplurality of substantially cylindrical micrografts 120 that can begathered in an exemplary dense arrangement, e.g., where adjacent ones ofthe exemplary micrografts 120 are in at least partially direct contacteach other. FIG. 4B is a schematic side view of the micrografts 120shown in FIG. 4A. This exemplary dense configuration can provide a graftthat is smaller than the overall area of the harvested donor site 100,but which can tend to heal faster and be less likely to produce visiblescarring than grafts formed using spaced-apart harvested tissue portions120, 320. Similar exemplary dense configurations of the harvested tissuecan be formed using, e.g., elongated strips of tissue 320 shown in FIG.3C or the like.

The exemplary biocompatible matrix 210 can be formed using one or morematerials structured to provide mechanical stability and/or support tothe harvested micrografts 200, and/or which may promote tissue regrowth.Examples of materials which can be used to form the matrix 210 caninclude polylactic acid (PLA), collagen (e.g., a collagen sponge), lowmelting agarose (LMA), hyaluronic acid (e.g., hyaluranon), ordevitalized animal or cadaveric skin. The matrix 210 can, for example,be formed of allogeneic skin that can be prepared, e.g., by freezing andthawing a portion of donor skin tissue several times. For example, aboutseven freezing/thawing cycles can be performed to effectively kill thecells in the donor skin for use as a matrix 210. The frozen and thawedtissue can then be washed with a detergent or other composition toremove dead cells, debris, etc.

In a further exemplary embodiment of the present disclosure, livingdonor skin tissue samples can be treated with radiation to form thematrix material 210. For example, donor skin tissue can be treated withlethal doses of x-rays, e.g. gamma rays, which can leave the cellsintact. Cells in the donor matrix material may thus stay alive for aparticular duration after radiation exposure, e.g., about 48-72 hours,before dying. Such a matrix 210 including short-lived cells caninitially support growth of implanted micrografts 120 but then die offbefore any significant interactions adverse to growth and/or viabilityof the micrografts 120 can occur such as, e.g., autoimmune responses.Alternatively, the implantation of such an irradiated matrix 210 withthe viable micrografts 120 can be delayed for a sufficient time afterirradiation, e.g., about 72 hours or more, to avoid any autoimmuneresponse between the micrografts 120 and the matrix 210. The matrix 210can also be prepared from donor skin, for example, by washing the cellsaway with detergent and acidic/basic solutions. An exemplary protocolfor preparing such matrices or scaffolds is described, e.g., in Alsberget al., Proc Natl Acad Sci USA. 2002 September 17; 99(19): 12025-12030.

In a further exemplary embodiment of the present disclosure, smallportions of a matrix material as described herein can be combined withthe micrografts 120, e.g., in a configuration similar to that shown inFIGS. 4A and 4B, except that some of the columns are formed of matrixmaterial. The micrografts 120 and matrix columns can be adhered using,e.g., adhesives, photochemical bonding, etc. The relative sizes andnumbers of the micrografts 120 and portions of matrix material can bevaried to generate a compound substance that has a particular fractionof micrograft material.

Nutrients or other additives can also be provided in the matrix 210 tofurther promote tissue regrowth. Such additives can include, e.g., oneor more growth factors, stem cells, etc. Examples of such growth factorsinclude, but are not limited to, vascular endothelial growth factor(VEGF), platelet-derived growth factor (PDGF), transforming growthfactor beta (TGF-β), and fibroblast, growth factor (FGF), which mayenhance or promote vascularization of the grafts. Epidermal growthfactor (EGF) and keratinocyte growth factors can also be used, and mayincrease mobilization and differentiation of certain skin cells, such askeratinocytes and fibroblasts. Platelet-rich plasma (which can beprepared, e.g., from a patient's own blood, using commercially availablesystems) can also be used to provide certain growth factors. Althoughsuch plasma can be more labor-intensive to prepare (from the standpointof clinical personnel) than exogenous growth factors, it may provide abetter approximation of the natural wound-healing environment. Suchgrowth factors can be introduced to the matrix 210, e.g. a LMA/collagenmix, at a moderate temperature where the matrix is still in a liquidform. The LMA/collagen matrix can then be formed by adjusting the pHvalue of the collagen solution before combining it with heated LMA. Fora solidification of the matrix 210, for example, the mix can be storedfor about 20 min at a temperature of about 37° C. before cooled it downto about 4° C.

Stem cell sources can include, e.g., adipose tissue-derived stem cellsand/or bone marrow-derived mesenchymal stent cells. Micrografts obtainedor harvested from skin tissue may also contain stem cells from hairfollicles. Such stem cells or other cells can be incorporated into thematrix by culturing the cells with the matrix prior to implantation ofmicrografts. Low-level light therapy (LLLT) can also be used tofacilitate growth and viability of the micrografts. For example, red ornear-infrared light can be used to illuminate the donor site and/or therecipient site after tissue harvesting and placement of the graft tissueto further promote healing and/or growth of the tissue.

Growth factors and/or other additives may be introduced into the matrix,e.g., by soaking the matrix material in a solution containing growthfactors prior to implantation of micrografts therein. The growth factorscan also be introduced by releasing them into the matrix over time usinga controlled-release mechanism, e.g., by directing or pumping the growthfactors from a reservoir into the matrix over time, or by embeddinggrowth factors in a biodegradable polymer that can be introduced intothe matrix. Certain growth factors or additives can also be attached tothe matrix material by chemical bonds that can be severed over time,thereby releasing the growth factors into the matrix gradually.

In certain exemplary embodiments of the present disclosure, certaintechniques such as photochemical tissue bonding can be used to improvemechanical stability of the micrografts 120 and/or tissue strips 320 inthe matrix 210. For example, a technique for a photochemical tissuebonding is described in U.S. Pat. No. 7,073,510. This technique includesan application of a photosensitizer to a tissue, followed by irradiationwith electromagnetic energy to produce a tissue seal. For example, aphoto sensitizer such as Rose Bengal can be applied to the matrix 210containing the exemplary micrografts 120 and/or tissue strips 320,followed by exposure of the matrix to green light for about two minutes.Photochemical tissue bonding can catalyze a polymerization reactionwhich may facilitate a stronger bonding of the micrografts 120 and/ortissue strips 320 to the matrix 210, where the matrix 210 can include aprotein such as, e.g., hyaluronic acid or collagen.

In further exemplary embodiments of the present disclosure, the matrixmaterial can be provided in a liquid or gel precursor form that can bemixed with the micrografts 120. The matrix material can then besolidified or gelled (e.g., either before or after introducing themixture of micrografts 120 and matrix material into the recipient site).Solidification of the matrix material to form the matrix 210 can beachieved using various procedures based on the matrix material used. Forexample, collagen gels (such as those depicted in FIG. 16) can beprovided in liquid form at lower temperatures, and may be solidified byraising the temperature to about 37° C. Other matrices 210 can be formedusing polymers that can be solidified by chemical cross-linkingmechanisms. Such mechanisms can be activated, catalyzed and/orinitiated, e.g., by temperature, light (similar to photochemical tissuebonding), a change in pH, or the like.

If the micrografts 120 are provided in the matrix 210 that is initiallyin a liquid form, the orientation of the micrografts 120 within thematrix 210 can be set and/or maintained in several ways. For example, alipid-rich material can be applied to the an upper end or portion of themicrograft 120 (e.g., the epidermal surface of the micrograft 120obtained from skin tissue), such that the tops of the micrografts 120would tend to float to the surface of the liquid matrix material,thereby aligning the upper ends of the micrografts 120 towards the uppersurface of the matrix 210. Alternatively, a metallic paint can beapplied to an upper portion of the micrografts 120, and a magnetic fieldcan then be applied proximal to the graft material to align themicrografts 120 within the matrix material. Such exemplary alignmentprocedures can be performed before and/or during solidification orgelation of the matrix material to produce a matrix 210 that hasmicrografts 120 therein that have a particular orientation.Alternatively, in certain exemplary embodiments of the presentdisclosure, the micrografts 120 can be provide in the matrix 210 withouta particular preferred orientation.

In still further exemplary embodiments of the present disclosure, anapparatus 500 can be provided, such as that shown in FIG. 5A, which canfacilitate harvesting of the exemplary micrografts 120 from the donorsite 100 as described herein. The exemplary apparatus 500 can include ahollow tube 510 that can be formed of metal or another structurallyrigid material. For example, the tube 510 can be formed using astainless steel tube, a biopsy needle, or a similar structure. The tube510 can be coated with a lubricant or low-friction material, such asTeflon®, to further facilitate the passage of the tubes 510 through thedonor site tissue 100.

The inner diameter of the tube 510 can be selected or structured toapproximately correspond to a particular diameter of a micrograft 120 tobe removed from the donor site 100, as described herein. According toone exemplary embodiment, the inner diameter of the tube 510 can be lessthan about 1 mm. For example, 18 or 20 gauge biopsy needles (e.g.,having an inner diameter of 0.838 mm and 0.564 mm, respectively) or thelike can be used to form the tube 510. A biopsy tube having a largergauge (and smaller inner diameter) can also be used. Based on theinteraction between the tube width or diameter of the harvestedmicrograft 120 can be slightly smaller than the inside diameter of theapparatus 500 used to harvest it.

A distal end of the tube 510 can be shaped to form a plurality of points520. For example, the two exemplary points or extensions 520 shown inFIG. 5A can be formed by grinding opposite sides of the tube 510 at anangle relative to the long axis of the tube 510. In a further exemplaryembodiment as shown in FIG. 5B, an exemplary apparatus 550 can beprovided that includes the tube 510 with three points or extensions 520provided at a distal end thereof. This exemplary configuration can beformed, e.g., by grinding 3 portions of the tube 510 at an anglerelative to the long axis thereof, where the three portions can bespaced apart by about 120 degrees around the perimeter of the tube 510.In still further exemplary embodiments, an apparatus can be provided forharvesting micrografts that includes a tube having more than threepoints or extensions 520 provided at a distal end thereof, e.g., a tube510 having four, five, six, seven or eight points 520.

The exemplary points or extensions 520 can facilitate insertion of theapparatus 500, 550 into the tissue at the donor site 100. The exemplarypoints or extensions 520 that can be formed, e.g., by grinding portionsof the distal end of the tube 510 can also have a beveled edge alongtheir sides, which can further facilitate insertion of the apparatus500, 550 into donor-site tissue. For example, the points or extensions520 that form a narrow angle at their tip can be inserted into thetissue using a smaller force as compared to points 520 having a largertip angle, although this force may be applied for a longer distanceand/or time to achieve full insertion of the tube 510 into the tissuethan that used for tips that have a wider tip angle and thus shorterlength of the angled tip region. The initial force needed for the tube510 to penetrate the tissue can be approximately proportional to thenumber of points 520 if the angle of each point or extension 520 is heldconstant. Providing a greater number of points extensions 520 at thedistal end of the tube can improve mechanical stability of the tube 510and/or geometrical control of the severed tissue, but it can use alarger force to penetrate the tissue.

The exemplary apparatus 500 can also included a collar or stop 540provided on an outer surface of the tube 510. The exemplary stop 540 canbe affixed to the tube 510 at a particular distance from the ends of thetips 520, or this distance may be adjustable, e.g., over a range oflengths by moving the stop 540 along the axis of the tube 510.

FIG. 6A illustrates the exemplary apparatus 500 after it is insertedinto the tissue at the donor site 100, e.g., until the stop 540 contactsthe surface of the donor site 100. A portion of the tissue 600 can bepresent within a lower portion of the tube 510. Lateral sides of thistissue portion 600 can be cut or severed from the surrounding tissue bythe distal end of the tube 510 and/or the points or extensions 520 asthe tube 510 penetrates into the donor site tissue 100. Such tissue 600can remain within the tube 510, and be separated from the donor site 100to form the micrograft 120, e.g., when the tube 510 is removed from thedonor site 100, as shown in FIG. 6B. The exemplary micrograft 120 thusformed can include both epidermal tissue 130 and dermal tissue 140.

The exemplary micrograft 120 can be removed from the apparatus 500,e.g., by providing pressure through an opening 620 at a proximal end ofthe tube 510 as shown, e.g., in FIG. 6C. Such pressure can bemechanical, hydraulic, pneumatic, etc. For example, the pressure can beprovided, e.g., by blowing into the opening, by squeezing a flexiblebulb attached thereto, by opening a valve leading from a source ofelevated pressure such as a small pump, etc. Alternatively, theexemplary micrografts 120 can be harvested by inserting the exemplaryapparatus 500 into a plurality of locations of the donor site 100. Eachmicrograft 120 within the tube 510 can then push any micrografts aboveit towards the opening 620. Once the tube 510 has been substantiallyfilled with the harvested tissue, each additional insertion of theexemplary apparatus 500 into the donor site 100 can facilitate pushingof an uppermost micrograft 120 within the tube 510 out of the proximalopening 620.

The exemplary apparatus 500 can be inserted into the donor site tissue100 to a depth corresponding approximately to a desired length of theharvested micrografts 120. Such distance can be determined and/orcontrolled, e.g., by appropriate placement or adjustment of the stop 540on the exemplary apparatus 500. For example, the exemplary apparatus 500can be configured or structured such that the points or extensions 520extend to a location at or proximal to the dermal/fatty layer junction610 as shown in FIG. 6A. For example, the micrograft 120 can be removedfrom the donor site 100 by removing the apparatus 500 from the donorsite without rotating the tube 510 around the axis thereof. In contrast,conventional biopsy needles and the like may require a rotation aroundthe long axis to facilitate removal of tissue samples from thesurrounding tissue. The points or extensions 520 provided on theexemplary apparatus 500 can facilitate such removal of the micrograft120 from the surrounding tissue at the donor site 100.

In certain exemplary embodiments of the present disclosure, some or allof the tissue at the donor site may be cooled, frozen, or partiallyfrozen prior to harvesting the micrografts 120. Such freezing mayfacilitate cutting, removal, handling, and/or viability of themicrografts 120. The donor site tissue 100 may be cooled or frozen usingconventional cooling techniques such as, e.g., applying a cryopspray orcontacting a surface of the donor site 100 with a cooled object for anappropriate duration. The exemplary apparatus 500 can also be cooledprior to harvesting the micrografts 120. Such cooling and/or freezingcan, e.g., increase mechanical stability of the micrografts 120 whenthey are harvested and/or placed in the matrix 210.

The exemplary micrografts 120 can be provided into the matrix 210 usingvarious techniques. For example, the individual micrografts 120 can beinserted into particular locations of the matrix 210 using, e.g.,tweezers or the like. The exemplary apparatus 500 containing a harvestedmicrograft 120, as shown in FIG. 6B, can also be inserted into alocation of the matrix 210, and pressure can be applied to the proximalopening 620 to push the micrograft 120 into the matrix 210. Theexemplary apparatus 500 can then be removed from the matrix 210, and theprocedure repeated to place a plurality of micrografts 120 in the matrix210. The proximal opening 620 can be covered while the apparatus 500 isbeing inserted into the matrix 210 to prevent the micrograft 120 frombeing pushed further up into the apparatus 500. For example, the upperportion of the tube 510 can be filled with a fluid, e.g., water or asaline solution, to provide an incompressible volume that can furtherprevent the micrograft 120 from rising further up into the tube 510.Such fluid can also facilitate a removal of the micrograft 120 from theexemplary apparatus 500 by providing pressure at the proximal opening620.

In a further exemplary embodiment of the present disclosure, anexemplary apparatus 700 can be provided, as shown in FIG. 7. Theapparatus 700 can include, e.g., a plurality of the tubes 510 affixed ormechanically coupled to a base 710. The tubes 510 can be provided invarious configurations, e.g., in a linear array, or in any one ofvarious two-dimensional patterns along the base 710. The number of thetubes 510 provided in the exemplary apparatus 700 can be, for example,at least 6 tubes 510, more than about 10 tubes 510, or more than about20 tubes 510. For example, the apparatus 700 can include a linear arrayof about 3 tubes 510, at least 6 tubes 510, or an array of about 12tubes 510, e.g., in a rectangular 3×4 array or a 2×6 array. Largerarrays of the tubes 510 can also be provided in further exemplaryembodiments of the present disclosure. The spacing of the tubes 510 canbe somewhat irregular or varied to avoid formation of recognizablepatterns in the donor and/or recipient sites. A larger number of thetubes 510 can be preferable to more rapidly harvest and/or implant alarge number of micrografts 120. However, a larger number of the tubes510 can also correspond to a greater force used to insert the tubes 510into tissue or a matrix simultaneously, which may be undesirable if theforce is too large. Also, the mechanical complexity of the apparatus 700can increase with a larger number of tubes 510.

A conduit 720 can be provided in communication with proximal openings620 of the tubes 510. The conduit 720 can also be provided incommunication, e.g., with a pressure source 730. For example, thepressure source 730 can include a pump or a deformable bulb or the like.The pressure source 730 can include, e.g., a flexible membrane providedin communication with the conduit 720, such that an elevated pressurecan be provided within the conduit 720 when the membrane is deformed.Such configurations can facilitate applying pressure to the proximalopenings 620 for removal and/or insertion of the micrografts 120 thatcan be harvested in the tubes 510, as described herein.

A vibrating arrangement 740 can optionally be provided in the apparatus700. The vibrating arrangement 740 can be mechanically coupled to thebase 710 and/or the tubes 510 to facilitate the insertion of the tubes510 into the tissue or matrix material for harvesting or placement ofmicrografts 120. The vibrating arrangement 740 can have an amplitude ofvibration in the range of about 50-500 μm, or between about 100-200 μm.The frequency of the induced vibrations can be between about 10 Hz andabout 10 kHz, or between about 500 Hz and about 2 kHz, or even about 1kHz. Particular vibration parameters can be selected based on, e.g., thesize, average spacing, and material of the tubes 510, the number oftubes 510 in the exemplary apparatus 700, and/or the tissue beingtreated. The vibrating arrangement 740 can include circuitry configuredto adjust the amplitude and/or frequency of the vibrations.

The exemplary apparatus 700 can be used to simultaneously obtain aplurality of the micrografts 120 in one or more of the tubes 510.Exemplary procedures for obtaining and removing such micrografts 120using the exemplary apparatus 700 can be similar to the proceduresdescribed herein for obtaining single micrografts 120 using theexemplary apparatus 500 shown in FIGS. 6A-6C.

A vibration can also assist in severing tissue proximal to the distalend of the tubes 510 after they are fully inserted into the donor site100. This can facilitate separation and/or extraction of the tissueportions within the tubes 510 from the donor site 100. These tissueportions can also be held by friction within the tubes 510 as the tubes510 are withdrawn from the donor site 100.

In further exemplary embodiments of the present disclosure, the donorsite tissue can be pre-cooled prior to insertion of the tubes 510, e.g.,using convective or conductive techniques such as applying a cryosprayor contacting the tissue surface with a cooled object. Cooling of thedonor site 100 can reduce a sensation of pain when the tubes 510 areinserted into the donor site tissue 100, and can also make the tissue100 more rigid and facilitate a more accurate severing of tissueportions (e.g., micrografts 120) by the tubes 510.

The positions and spacing of the tubes 510 in the exemplary apparatus700 can be determined, e.g., based on characteristics of the micrografts120 to be obtained, a damage pattern to the donor site 100, and/or otherfactors as described herein above. The number of the tubes 510 providedin the exemplary apparatus 700 can be selected based on various factors.For example, a larger number of tubes 510 may be desirable to allow moremicrografts 120 to be harvested simultaneously from a donor site 100.Such exemplary configuration can facilitate a more efficient harvestingprocess. A smaller number of the tubes 510 can be easer to insertsimultaneously into the donor site tissue 100. Further, the exemplaryapparatus 500 having a very large number of the tubes 510 can bechallenging to manufacture and/or maintain.

The harvested tissue portions can be deposited directly from the tubes510 into the biocompatible matrix material 210. The tubes 510 and thetissue portions contained therein can be cooled before removal of thetissue portions. This can stiffen the tissue portions within the tubes510 and make them easier to manipulate and position.

In a further exemplary embodiment of the present disclosure, anapparatus can be provided that includes a plurality of substantiallyparallel blades. The ends of certain ones of the adjacent blades can beconnected or closed off to provide, e.g., narrow rectangular openingsbetween adjacent blades. Such an exemplary apparatus can be used, e.g.,to form the tissue strips 320, such as that shown in FIG. 3C. Spacings,lengths, and other features of this exemplary apparatus can be selectedbased on factors similar to those described herein, e.g., for theexemplary apparati 500, 700.

In further exemplary embodiments of the present disclosure, theexemplary methods and apparati described herein can be applied to othertissues besides skin tissue, e.g., internal organs such as a liver orheart, and the like. Thus, grafts can be formed for a variety of tissueswhile producing little damage to a donor site and facilitating rapidhealing thereof, while creating graft tissue suitable for placement atrecipient sites.

An image of a distal end of an exemplary apparatus that includes twopoints is shown in FIG. 8A. This exemplary apparatus is similar to theexemplary apparatus 500 illustrated, e.g., in FIG. 5A. A further rotatedimage of this exemplary apparatus is shown in FIG. 8B. Such exemplaryapparatus was formed using a tube having an outside diameter of about 1mm, and an inside diameter of about 0.5 mm. The points or extensionswere formed by grinding two opposite sides of the distal end of the tubeat an appropriate angle relative to the axis of the tube. The angle usedin the exemplary apparatus shown in FIGS. 8A and 8B was about 30degrees, although other angles may also be used. A beveled edge of thetube wall can be seen along the sides of the points or extensions. Theshape of these points can facilitate insertion of the apparatus intotissue of a donor site and/or separation of a portion of micrografttissue from the donor site, as described in more detail herein. Forexample, such micrografts can be separated and removed from the donorsite by inserting and withdrawing the apparatus from the donor sitetissue without rotating the tube along its axis.

The geometry of the distal (‘piercing’) end of an exemplary tube 510that includes two points 520 can be characterized by an angle α, whichrepresents the angle between each of the opposing lateral sides of thetube 510 that form the points or extensions 520 and the longitudinalaxis of the tube 510. The angled lateral sides at the distal end of thetube 510 can each be ground or cut at this angle α relative to the axisof the tube 510, e.g., to form a beveled structure at the distal end ofthe tube 510. The angle formed at the points or extensions 520 whenviewed from the side can thereby be represented by the angle 2α, asshown in FIG. 8C. For example, the exemplary tip angle of about 30degrees shown in FIGS. 8A and 8B corresponds to an angle α of about 15degrees.

A further beveled surface can be optionally provided in a directionorthogonal to the primary bevel that is characterized by the angle αshown in FIG. 8C. This second bevel can be characterized by an angle β,which represents the angle at which each of the opposing lateral sidesof the tube 510 can be ground or cut relative to the longitudinal axisof the tube 510, as shown in FIG. 8D. This second bevel can be providedto reduce the size or width of the sharp edge of the tips 520 formed atthe end of the tube 510.

FIGS. 8E, 8F and 8G illustrate exemplary beveled ends of the tube 510,where the primary bevel angle α can be about 6 degrees. For example,there is no secondary bevel formed in FIG. 8E, and the points orextensions 520 have a flat cutting edge with a length equal to thethickness of the wall of the tube 510. FIGS. 8F and 8G include asecondary bevel, where the angle β corresponding to this secondary bevelis also 6 degrees. The secondary bevel is relatively shallow in theexemplary tip region shown in FIG. 8F, which provides a shorter flatcutting edge at the tips of the points or extensions 520. The secondarybevel is deeper in the exemplary tip region shown in FIG. 8G, such thatthe tips of the points or extensions 520 form sharp points that canfacilitate penetration of tissue more easily.

FIGS. 8H, 8I, and 8J illustrate exemplary beveled ends of the tube 510,similar to those shown in FIGS. 8E-8G, but where the primary bevel angleα is 12 degrees. There is no secondary bevel formed in FIG. 8H, ashallow secondary bevel shown in FIG. 8I having an angle β of about 6degrees, and a deep secondary bevel shown in FIG. 8J, again with anangle β of, e.g., about 6 degrees.

The various geometries and points 520 described above and shown in FIGS.8A-8F can be used in any of the exemplary embodiments of the presentdisclosure, e.g., for the various devices and methods described herein.For example, the hollow tube 510 can be provided with a bevel angle α ofless than about 15 degrees, e.g., about 12 degrees. Such an acute tipangle can provide sharp tips of the points or extensions 520 that caneasily penetrate a biological tissue or matrix material. A narrower tipangle α, e.g. about 6 degrees as shown in FIGS. 8E-8G, may be preferablefor harvesting and/or implanting micrografts in a denser or toughertissue or matrix material, where the narrower tips of the points orextensions 520 can be configured to more easily cut through the tissueor matrix material when the tube 510 is inserted therein. A secondarybevel having an angle β, such as the exemplary tips of the points orextensions 520 shown in FIGS. 8F, 8G, 8I, and 8J, can further facilitateinsertion of the distal end of the tube 510 into various materials byproviding the tips of the points or extensions 520 that are smaller andmore pointed. However, the tips of the points or extensions 520 that aresharper and/or narrower, e.g., those having small tip angle α and/or asecondary bevel with angle β, can also be more prone to wear, bending,or other deformation if the tube 510 is repeatedly inserted into tissueor a matrix. Accordingly, the tip geometry selected for a particularapplication can be selected based on the type of material or tissue theapparatus will be used with, as well as the desired lifetime of the tube510.

FIG. 9 shows an exemplary image of a plurality of micrografts obtainedfrom a donor site of ex vivo skin tissue using the exemplary apparatusshown in FIGS. 8A-8B. The micrografts are elongated and substantiallysimilar in shape, although details of the shapes may be somewhatirregular. An upper portion of these micrografts includes epidermaltissue, and the lower portion of these micrografts include dermal tissueremoved from the donor site. The width of these micrografts is slightlysmaller than the internal diameter of the tube 510 shown in FIGS. 8A-8Bthat was used to harvest them.

The micrografts shown in FIG. 9 were removed from the apparatus byinserting the exemplary apparatus into donor site a plurality of times,until the tube was filled with harvested tissue. Each subsequentinsertion of the apparatus into the donor site tissue then forced theuppermost micrograft out of the proximal end of the tube, where it wasretrieved individually for analysis. Such micrografts can also beremoved by applying pressure to the proximal end of the tube containingthe micrograft, to expel it from the distal end of the tube as describedherein.

In still further exemplary embodiments of the present disclosure, anapparatus 1000 can be provided, such as that shown in FIG. 10A, whichcan facilitate harvesting of the exemplary micrografts 120 from thedonor site 100 and optionally placing them in a biocompatible matrix210, as described herein below. The exemplary apparatus 1000 can includea hollow tube 1010 that can include a plurality of points 1020 at thedistal end thereof as described herein, which can be similar to thehollow tube 510 shown in FIGS. 5A and 5B.

In one exemplary embodiment of the present disclosure, the tube 1010 canbe provided with two points 1020, and the points 1020 can be formed bygrinding opposite sides of the distal end of the hollow tube 1010 at anacute angle, e.g., about six degrees, relative to the longitudinal axisof the tube 1010. Such an acute tip angle, e.g., of about 12 degrees orless, can be particularly effective for penetrating and cutting thebiological tissue to remove small micrografts 120 therefrom. Such a tubeprovided with two points 1020 can use a force approximately twice thatassociated with a single-point needle of similar diameter to penetratetissue or another material.

For example, FIG. 19A illustrates an exemplary sequence of inserting andextracting or withdrawing a harvesting needle device from a tissuelayer. The exemplary needle device can be similar to the hollow pointedtube 510 shown in FIG. 5A, or the exemplary pointed tube 1010 shown inFIG. 10A. A plot of the position of the needle relative to the tissue asa function of time is shown in FIG. 19B, together with a plot of thecorresponding force applied to the exemplary needle (e.g., along thelongitudinal axis) to perform the insertion and extraction sequence. Theneedle used to obtain the data shown in FIG. 19B was formed from a 25gauge tube with a tip bevel angle α of 6 degrees and a side bevel angleβ of 6 degrees, similar to the exemplary needle geometry shown in FIGS.8C, 8D, and 8F.

As provided in the graph of FIG. 19B, the needle can be rapidly insertedthrough the tissue layer at about 6 seconds and held there for about onesecond, and then steadily withdrawn to a position outside of the tissuelayer over a time span of about 0.8 seconds. There can be an apparentdelay in the force needed to maintain the needle at a certain depthafter insertion into the tissue, and a residual force on the needleafter it is brought to a position outside of the tissue layer. Duringinsertion and extraction of the needle, which was formed using a 25gauge tube, the maximum and minimum forces observed on the needle wereabout −0.2 N and 0.2 N, respectively. The delay between movement of theneedle in and out of the tissue and the resultant force on the needlemay result at least in part from deformation of the tissue as the needleis inserted and withdrawn, and/or some adhesion of the tissue to theneedle as the needle is moved relative to the tissue. The small forceappearing at the beginning and end of the sequence plotted in FIG. 19Bare likely associated with some component friction and/or the verticalload cell sensing attached components.

In one exemplary embodiment of the present disclosure, the tube 1010 canbe formed using a 25 gauge thin-wall needle, with an exemplary outerdiameter of about 0.51 mm and an internal diameter of about 0.31 mm.This exemplary needle size can be used to harvest micrografts 120 havinga width or diameter of about 0.2 mm. The tube 1010 can be formed of anysufficiently strong material that is preferably biocompatible or inertwith respect to biological tissue, e.g., a 304 stainless steel, asurgical stainless steel, etc. Further finishing processes can beapplied to the tube 1010, such as electropolishing to increase sharpnessof the cutting edges or providing a ME-92® chromium coating to increasethe material strength. Such finishing processes can increase the cuttingeffectiveness and/or improve the useful service life of the needle 1010.

The tube 1010 can be slidably attached to a substrate 1030, such thatthe tube 1010 passes through a hole provided in the substrate 1030, asshown in FIG. 10A. The position of the tube 1010 relative to thesubstrate 1030 can be controlled by a positioning arrangement that cancontrollably translate the tube 1010 relative to the substrate 1030,e.g., substantially along the longitudinal axis of the tube 1010. Inthis manner, the distance that the distal end of the tube 1010 protrudespast the lower surface of the substrate 1030 can be controllably varied.

The exemplary apparatus 1000 further includes a pin 1040 provided in thecentral lumen or opening of the tube 1010. The diameter of the pin 1040can be substantially the same as the inner diameter of the tube 1010 orslightly smaller, such that the pin 1040 can be translated along theaxis of the tube 1010 while filling or occluding most or all of theinner lumen of the tube 1010. The pin 1040 can be formed of alow-friction material, or coated with a low-friction material such as,e.g., Teflon® or the like, to facilitate motion of the pin 1040 withinthe tube 1010 and/or inhibit accumulation or sticking of biologicalmaterial to the pin 1040. The distal end of the pin 1040 can besubstantially flat to facilitate displacement of a micrograft 120 withinthe tube 1010 when the pin 1040 is translated.

In one exemplary embodiment of the present disclosure, the tube 1010 canbe formed using a 25 gauge thin-wall needle, with an exemplary outerdiameter of about 0.51 mm and an internal diameter of about 0.31 mm.This exemplary needle size can be used to harvest micrografts 120 havinga width or diameter of about 0.2 mm. A pin 1040 that can be used with atube 1010 of this size can have an outer diameter of about 0.24 mm. Thedifference between the inner diameter of the tube 1010 and the diameterof the pin 1040 can facilitate motion of the pin 1040 within the tube1010, whereas the pin is sufficiently wide to push a micrograft 120 outfrom the interior of the tube 1010. The tube 1010 and/or the pin 1040can be formed of any sufficiently strong material that is preferablybiocompatible or inert with respect to biological tissue, e.g., a 304stainless steel, a surgical stainless steel, etc.

The pin 1040 can be provided with a further positioning arrangement thatcan controllably translate the pin 1040 relative to the tube 1010 e.g.,substantially along the longitudinal axis of the tube 1010. In thismanner, the position of the distal end of the tube 1010 relative to thatof the distal end of the pin 1040 can be controllably varied. Forexample, the location of the distal ends of both the tube 1010 and thepin 1040 relative to that of the lower surface of the substrate 1030 canpreferably be controllably and independently selected and varied.

An exemplary illustration of the tube 1010 and pin 1040 as describedherein is shown in FIG. 10B, which shows the pin 1040 positionedrelative to the tube 1010 such that their distal ends are substantiallyaligned. Portions of the pin 1040 and/or tube 1010 can optionally beprovided with a coating or surface treatment to reduce friction betweenthem and/or between either component and biological tissue. Exemplarycoatings that can be used include a plastic or polymer, e.g. nylon orpolyethylene, a polished metal alloy, or the like.

A schematic side view of an exemplary apparatus 1100 that can be used tofacilitate harvesting of the exemplary micrografts 120 from the donorsite 100 and optionally placing them in a biocompatible matrix 210 isshown in FIG. 11. The apparatus 1100 can include the tube 1010, pin1040, and substrate 1030 shown in FIG. 10A. A plate 1110 or othersupporting structure can be affixed to the substrate 1030, or optionallyprovided with the substrate 1030 as a single unitary component.

A first actuator 1120 can be affixed to the plate 1110, and mechanicallycoupled to the tube 1010 using a first coupling arm 1125 or the like.Similarly, a second actuator 1130 can be affixed to the plate 1110, andmechanically coupled to the pin 1040 using a second coupling arm 1135 orthe like. The first actuator 1120 and second actuator 1130 can beconventional linear actuators or the like. Such actuators 1120, 1130 caninclude appropriate controls such that the locations of the couplingarms 1125, 1135 relative to the plate 1110 (and substrate 1130) can becontrollably positioned and varied independently. For example, thelinear range of motion of the actuators 1120, 1130 can be about 2 cm orless for many typical uses of the apparatus 1100, e.g., for harvestingmicrografts of skin tissue and placing them in a matrix as describedherein below.

In the exemplary schematic configuration of the apparatus 1100 shown inFIG. 11, the translation or positioning range of the actuator 1120 canbe selected such that the distal end of the tube 1010 can be raisedabove the lower surface of the substrate 1030, and lowered such that thedistal end protrudes to a maximum distance of about 1-2 cm below thelower surface of the substrate 1030. Similarly, the positioning range ofthe actuator 1130 can be selected such that the distal end of the pin1040 can be raised above the distal end of the tube 1010 by a distanceof about 1-2 cm, and lowered such that the distal end of the pin 1040 issubstantially aligned with the distal end of the tube 1010 (e.g.,aligned with the tips of the points 1020). The translation range of theactuators 1120, 1130 can also be greater than these exemplary distances,for example, if the donor tissue being harvested is relatively thick.Alternatively, these translation ranges may be somewhat smaller ifmicrografts are being harvested from thin layers of tissue andoptionally placed in a thin matrix.

An exemplary sequence for harvesting a micrograft 120 from a donor site100 and placing it in a matrix 210 in accordance with embodiments of thepresent disclosure is shown in FIGS. 12A-12E. As an initial matter, thesurface of the donor site can be cleaned, sterilized, shaved, and/orotherwise prepared, and a portion or sheet of a matrix material 210 canbe placed thereon, as shown in FIG. 12A. The distal ends of the tube1010 and the pin 1040 can be aligned by the respective actuators (notshown), and positioned proximal to the upper surface of the matrix 210.The tube 1010 and the pin 1040 can be translated such that the distalends thereof penetrate or pass through the thickness of the matrix 210,and are positioned proximal to the surface of the donor site 100 asshown in FIG. 12B. The tube 1010 and pin 1040 can push aside a smallamount of the matrix material, which can be viscous, pliable, elastic,or the like, as they pass through the matrix 210.

The tube 1010 can then be translated downward such that it penetratesinto the tissue of the donor site 100, while the position of the pin1040 can be held substantially stationary relative to the donor site 100such that its distal end remains positioned proximal to the surface ofthe donor site 100, as shown in FIG. 12C. The distal end of the tube1010 can sever a portion of the tissue from the surrounding tissue atthe donor site as the tube 1010 penetrates the donor site, such that aportion of tissue from the donor site 100 can be located within thedistal end of the lumen of the tube 1010.

The depth of penetration of the tube 1010 into the donor site 100 can becontrolled by the tube actuator 1120, as shown in FIG. 11. The tube 1010can be translated such that the distal end thereof is located at aparticular depth within the donor site tissue. For example, if the donorsite is skin tissue, the distal end of the tube 1010 can be extendedsuch that it is proximal to the lower end of the dermis layer, e.g.,such that it penetrates slightly into the underlying fatty tissue asshown in FIG. 12C.

The tube 101 and pin 1040 can then be lifted or retracted simultaneouslysuch that they substantially maintain their relative positions until thedistal end of the tube 1010 is proximal to the surface of the donor site100 or slightly above it, as shown in FIG. 12D. The portion of tissuesevered from the donor site 100 by the tube 1010, which can be used as amicrograft 120, can also be held within the distal end of the tube 1010,such that it is lifted or removed from the donor site 100 as the tube1010 is withdrawn therefrom. If the tissue being harvested is skintissue, removal of the micrograft 120 from the donor site can befacilitated if the tube 1010 first penetrates to at least an uppersurface of the subdermal fatty layer. The lower end of the micrograft120 can be more easily detached or torn away from adjacent fatty tissuethan from dermal tissue.

As shown in FIG. 12D, s withdrawal of the tube 1010 and the pin 1040together from the donor site 100 can place the micrograft 120 within thelayer of the matrix material 210. For example, the tube 1010 and the pin1040 can be raised substantially simultaneously such that the distal endof the tube 1010 is positioned proximal to the lower surface of thematrix material 210 or within the matrix material 210. The tube 1010 canthen be further retracted from the matrix 210 while holding the locationof the pin 1040 substantially stationary relative to the matrix 210, asshown in FIG. 12E. This exemplary procedure facilitates placement of themicrograft 120 substantially within the material of the matrix 210 usingthe pin 1040 as the tube 1010 is retracted from around the micrograft120. In this manner, the micrograft 120 can be placed within the matrix210 and remain there, while the tube 1010 and the pin 1040 arecompletely removed from the donor site 100 and matrix 210.

The exemplary apparatus shown in FIG. 11 and the tissue harvestingsequence shown in FIGS. 12A-12D and described herein provide severaladvantages for harvesting micrografts 120 from the donor site andplacing them in the matrix 210 to facilitate their use in grafting orautografting procedures. For example, the micrograft 120 can bepositioned within the matrix 210 without being exposed to open air (oranother intermediate environment) to reduce a chance of contamination,biological stress, etc. The various penetration depths can be selectedor adjusted based on the desired depth of the micrografts to beharvested, the thickness of the matrix material 210 to be used, etc. Themicrografts 120, which may be difficult to manipulate because of theirsmall size and/or soft tissue consistency, can be placed in the matrix210 in substantially the same orientation as they had within the donorsite.

The substrate 1030 shown in FIG. 11, if present, can provide amechanical stabilization to the matrix 210 and a surface of the donorsite 100 when the apparatus 1100 is placed over the layer of matrixmaterial 210. Placing the substrate 1030 over the matrix 210, ifpresent, and/or the donor site 100 can inhibit motion and/or deformationof the matrix 210 and tissue of the donor site 100 while the micrografts120 are being harvested.

In a further exemplary embodiment of the present disclosure, thesubstrate 1310 can include a plurality of strips or slats 1315, as shownin FIG. 13B, with elongated openings between them. The black dots shownin FIG. 13B are micrografts that were harvested from a donor site thatwas colored black, and implanted into the recipient site shown in thisfigure. Both the donor and recipient sites were skin tissue of a pig,creating an allograft. The substrate 1030 can be placed over the donortissue 100 as described above to stabilize the tissue 100, while theharvesting tubes 510, 1010 can be inserted into the tissue 100 betweenthe slats 1315. The substrate 1310 can also be used to facilitatepositioning of the tubes 510, 1010 relative to the donor tissue, e.g.,for repeated insertions and extractions of the tube 510, 1010 from aregion of donor tissue 100. The substrate 1030 can also be provided withother geometrical shapes and arrangements of openings therethrough, suchas a plurality of openings having an elliptical, triangular, square, orother shape, or a combination thereof.

In a further exemplary embodiment of the present, disclosure, thesubstrate 1030 can be placed directly on the surface of the donor site100 to provide mechanical stabilization and reduce movement of thetissue of the donor site 100, as shown in FIG. 13. The substrate 1030can be provided with one or more holes 1310 therethrough. One or more ofthe tubes 1010 can be configured to pass through the holes 1310 to severand extract micrografts 120 from the donor site, e.g., as describedabove and illustrated in FIGS. 12A-12E. The matrix 210 can be providedon top of the substrate 1030 as shown in FIG. 13A. Optionally, a layerof dressing material 1320 or the like can be placed on the top surfaceof the matrix 210 to further stabilize the matrix 210 and/or facilitatehandling of the matrix 210. If the substrate 1030 is provided betweenthe matrix 210 and the donor site, as shown in FIG. 13A, the translationdistances or heights of the tube 1010 and pin 1030 shown in the sequenceof FIGS. 12A-E can be controlled appropriately such that the micrografts120 are harvested from the donor site 100 and deposited in the matrix210 above the substrate 1030.

In a further exemplary embodiment of the present disclosure, twosubstrates 1400, 1410 can be used to provide further stabilization, asshown in FIG. 14. A lower substrate 1410 having one or more holes 1420therethrough can be provided on the surface of the donor site 100,similar to the substrate 1030 shown in FIG. 13A. The matrix 210 can beprovided on top of the lower substrate 141., An upper substrate 1400 canbe provided on top of the matrix 210 or dressing material 1320, ifpresent. Such upper substrate 1400 can be provided with one or moreholes 1425 therethrough, which can correspond to and/or be aligned withthe holes 1420 provided in the lower substrate 1410. These aligned holes1420, 1425 can facilitate translation of the tube 1010 into and out ofthe matrix 210 and donor site tissue 100 while providing mechanicalstability to both the donor site 100 and the matrix 210. Such alignedholes 1420, 1425 in the lower substrate 1410 and upper substrate 1400can also provide a further mechanical stability and an improvedalignment of one or more of the tubes 1010 as they are translatedvertically through the holes 1420, 1425.

In a still further exemplary embodiment of the present disclosure, abarrier 1430, e.g. a sidewall or the like, can be provided between thelower substrate 1410 and the upper substrate 1400 as shown in FIG. 14,e.g., proximal to the perimeter of one or both substrates. The lowersubstrate 1410, the upper substrate 1400, and the barrier 1430 cantogether form an enclosure around the matrix 210. This exemplaryconfiguration can facilitate containment of the matrix 210, e.g., if thematrix 210 is formed from a viscous or easily deformable material.Accordingly, the exemplary embodiment of the present disclosure shown inFIG. 14 can facilitate a placement of micrografts 120 into the matrix210 that may likely not be mechanically rigid or stable. The exemplaryembodiment shown in FIG. 14 may be used with other various embodimentsof the present disclosure described herein.

The exemplary apparatus 1100 can be used to harvest a plurality ofmicrografts 120 from the donor site 100, and optionally place them inthe matrix 210. For example, the substrate 1030 can be provided with aplurality of spaced-apart holes therethrough. The plate 1110 shown inFIG. 11, or another portion of the apparatus 1100 supporting the tube1010 and pin 1040, can be configured or structured to be translatableover the substrate 1030, such that the tube 1010 and pin 1040 can bepositioned over a plurality of holes in the substrate 1030. Suchtranslation can be in one dimension (e.g., linear) or in two dimensions,e.g., over a particular surface region of the substrate 1030. In thisexemplary manner, a plurality of micrografts 120 can be harvested from adonor site 100 and placed in a plurality of locations in the matrix 210,e.g., proximal to a plurality of the holes in the substrate 1030, whilemaintaining the substrate 1030 and the exemplary apparatus 1100 in asingle location relative to the donor site 100.

In a further exemplary embodiment of the present disclosure, theapparatus 1100 shown in FIG. 11 can be provided with a plurality of thetubes 1010 and of the pins 1040. All of the tubes 1010 can betranslatable together using a single first actuator 1120, or certainones of the tubes 1010 can be translated simultaneously and/orsequentially using a plurality of the first actuators 1120 andappropriate first coupling arms 1125. Similarly, the plurality of pins1040 can be translatable together using a single second actuator 1130,or certain ones of the pins 1040 can be translated simultaneously and/orsequentially using a plurality of second actuators 1130 and appropriatesecond coupling arms 1135. In general, it may be preferable for thetranslation of each of the tubes 1010 to be coordinated with itsassociated pin 1040 (i.e., the pin 1040 provided in the lumen of theparticular tube 1010). In this exemplary manner, each combination of thetube 1010 and the associated pin 1040 can be controlled to perform theexemplary harvesting and implanting sequence illustrated in FIGS.12A-12E.

The matrix 210 containing one or more micrografts 120 as describedherein can be used as a graft material that can be placed over arecipient site of damaged tissue that has been suitably prepared. Ingeneral, this graft material will include a plurality of micrografts 120that are provided in the matrix 210. The spacing of the micrografts 120in the matrix 210 can be selected to facilitate the micrografts to growwithin or through the matrix 120 and eventually provide sufficientcoverage and/or repair of the damaged region. The micrografts 120 can beplaced in the matrix 210 in a uniform pattern, randomly, or in any otherdesired spatial arrangement. In certain exemplary embodiments of thepresent disclosure, the density or spacing of the implanted micrografts120 can vary in different regions of the matrix 210. For example, ahigher density and/or smaller spacing of the micrografts 120 can beprovided closer to the edges of the matrix 210 to improve peripheralintegration and/or revascularization of the graft. A sterile dressing orthe like can be placed over the graft material after it is placed on thedamaged region of tissue. Such dressing can be adhered to the graftmaterial to facilitate handling and positioning of the graft material onthe recipient site.

In a further exemplary embodiment of the present disclosure, theharvested micrografts 120 can be introduced or transplanted directlyinto, e.g., substantially whole tissue at the recipient site. Forexample, the micrografts 120 can be harvested from the donor site 100that may contain melanocytes, and inserted directly into tissue at arecipient site that lacks sufficient melanocytes. Such exemplaryprocedure can be used to repigment skin tissue, e.g., to treat vitiligoor similar conditions. Tissue at the recipient site 100 can also befrozen or partially frozen, as described herein, prior to insertion ofmicrografts 120 therein.

An exemplary apparatus 1500 for implanting micrografts 120 into arecipient site 1510 is shown in FIGS. 15A-15E. The apparatus 1500 caninclude the hollow tube 1010 that can include a plurality of the points1020 at the distal end thereof, and the pin 1040 provided in the centrallumen or opening of the tube 1010, similar to the apparatus shown inFIG. 10A. The exemplary apparatus 1500 can further include a hollowpiercing needle 1520 that can be provided around the tube 1010, as shownin FIG. 15, such that the tube 1010 can be advanced and/or retractedwithin the piercing needle 1520. The piercing needle 1520 can include asingle point 1525 configured to pierce and penetrate a biologicaltissue. The piercing needle 1520 can be manually controlled, or it canbe controlled using an actuator, e.g., similar to the actuators 1120,1130 in the exemplary apparatus 1100 shown in FIG. 11.

In an exemplary method of the present disclosure, the tube 1010 and thepin 1040 can be used to harvest a tissue micrograft 120, for example,using the exemplary harvesting sequence shown in FIGS. 12A-D. Themicrograft 120 can be retained in the tube 1010 when the tube 1010 andpin 1040 are fully withdrawn from the donor site 100. The tube 1010 andthe pin 1040 can be located within the piercing tube 1520 such that thedistal end of the tube 1010 is within the piercing needle 1520, as shownin FIG. 15A.

The piercing needle 1520 can then be advanced into the recipient site1510, such that the point 1525 of the piercing needle 1520 separates aportion of the tissue in the recipient site 1510 as shown in FIG. 15B.The tube 1010 and pin 1040, together with the micrograft 120, can beadvanced forward together with the piercing needle 1520 such that thedistal end of the tube 1010 containing the micrograft 120 is locatedbelow or proximal to the surface of the recipient site 1510, as shown inFIG. 15B. The piercing needle 1520 can be withdrawn from the recipientsite 1510 while holding the tube 1010 substantially stationary relativeto the recipient site 1510, such that the distal end of the tube 1010containing the micrograft 120 is located within the separated tissue ofthe recipient site 1510, as shown in FIG. 15C.

The tube 1010 can then be withdrawn from the recipient site 1510 whileholding the pin 1040 substantially stationary relative to the recipientsite 1510, such that the micrograft 120 remains within the separatedtissue of the recipient site 1510 when the tube 1010 is withdrawn, asshown in FIG. 15D. Upon removal of the apparatus 1500 from the recipientsite 1510, the micrograft 120 can remain within the recipient site 1510in a known orientation, as shown in FIG. 15E.

Such direct implantation can be used for tissue normalization, e.g., totreat vitiligo by transplanting the micrografts 120 containing melanindirectly into a depigmented recipient site 220. The exemplarymicrografts 120 can also be harvested from a healthy donor site 100 andplaced directly into the recipient site 1510 that includes scar tissueto facilitate growth of healthy tissue in the scar using the exemplarymethod and apparatus shown in FIGS. 15A-5E. In a further exemplaryembodiment of the present disclosure, portions of tissue can be removedfrom the recipient site 1510 prior to placing micrografts 120 in holesthat are formed at the recipient site 1510 by the removal of thesetissue portions. The holes can be about the same size or slightly largerthan the size of the micrografts 120 to be inserted therein, tofacilitate such insertion. The holes can be formed at the recipientsite, e.g., using one or more of the tubes 510 as described herein, byremoving or ablating the tissue using, e.g., an ablative laser, etc.

The exemplary method and apparatus illustrated in FIGS. 15A-15D can beused for a variety of procedures, including grafting between or amongvarious tissues besides skin, such as, e.g., muscle tissue, organtissue, etc. “Hybrid” or heterogeneous grafts involving differenttissues can also be generated using the exemplary methods and apparatusdescribed herein. For example, the micrografts 120 from the donor sitehaving a first tissue type can be placed in a second type of tissue at adonor site. Such exemplary grafting procedures can be used in manydifferent applications. For example, micrografts 120 from an endocrineorgan can be placed in a donor site 1510 that includes skin. Forexample, pancreatic tissue micrografts can be placed in skin tissue toprovide for insulin secretion. As another example, smooth muscle tissuecan be introduced into the gastrointestinal tract. Micrografts 120obtained from other functional tissues may be placed in donor siteshaving different characteristics.

In certain exemplary embodiments of the present disclosure, the piercingneedle 1520 can be provided around at least a portion of the tube 510,1010 without a sharpened point 1525. The distal end of the piercingneedle 1520 can be flat, for example, and the outside of the distal endmay optionally be widened or flanged. Such a piercing needle 1520 canserve as a guide and/or support for the tube 510, 1010, and can reduceor prevent bending, distortion, breakage, etc. of the tube 510, 1010when it is inserted into and withdrawn from a donor tissue site. It canalso facilitate control of the insertion depth of the tube 1010 and/orthe pin 1040 (if present) in the tissue.

In further exemplary embodiments of the present disclosure, a conduitcan be provided in communication with the lumen of the tubes 510, 1010described herein, e.g., connected to a proximal end of the tubes 510,1010. Such conduit can be configured similar to the conduit 720 shown inFIG. 7. The conduit can also be provided in communication with a sourceof low and/or high pressure, e.g., a vacuum arrangement and/or a sourceof pressurized gas or liquid. For example, the diameter of the pin 1040can be sized slightly smaller than the internal lumen diameter of thetube 1010. Such a configuration can facilitate a fluid such as a gas topass through the lumen of the tube 1010 around the pin 1040, and furtherallow pressure differentials to propagate through the lumen of the tube1010. A controlled application of low pressure from the conduit to thelumen of the tube 1010 can facilitate a separation of the micrografts120 from surrounding tissue. Similarly, an application of an elevatedpressure from the conduit into the lumen of the tube 1010 can facilitateremoval or expulsion of a micrograft 120 from the tube 1010 after it isharvested.

In a further exemplary embodiment of the present disclosure, a fluid canbe provided within the tube 510 shown in FIGS. 5A and 5B or the tube1010 shown in FIG. 10A, e.g., such that a portion of the fluid ispresent between the tube 1010 and the pin 1040. This fluid can reducefriction between the pin 1040 and the tube 1010. The fluid can alsoreduce or prevent a buildup or accumulation of biological tissueproximal to the distal end of the tube 1010 when the tube 1010 is usedto harvest a plurality of tissue micrografts 120. The fluid can alsofacilitate a retention of the micrografts 120 in the tube 1010 and/orrelease of the micrografts 110 from the tube, e.g., by providing thefluid at a reduced or elevated pressure during appropriate steps in theexemplary micrograft manipulation sequences shown, e.g., in FIGS. 6A-6C,12A-12E, and/or 15A-15D. For example, the fluid can improve the accuracyof placement of the micrografts 120 in tissue 1510 at the donor site orin the matrix 210 as described herein. Such accuracy in positioning theharvested micrografts 120 can, e.g., reduce or prevent a formation ofcysts or other undesirable results when the micrografts 120 are allowedto grow after harvesting.

Such fluid can be provided, e.g., through a conduit or the like that canbe provided in communication with a proximal end of the tube 1010,similar to the conduit 720 shown in FIG. 7. Alternatively, the fluid canbe provided through an opening formed in the side of the tube 1010, etc.If the piercing needle 1520 is used, such as provided in the exemplaryapparatus 1500 shown in FIG. 15, the fluid can also or alternatively beprovided between the piercing needle 1520 and the tube 1010.

Exemplary fluids that can be used can be biocompatible, inert withrespect to the biological tissue, etc. Such fluid preferably would notproduce any adverse effect when contacting the tissue 100, 1510. Forexample, the fluid may include a saline solution, glycerol, or the like.It may be buffered, and can include one or more additional components,such as anticoagulants, antibacterial agents, coagulants, etc. One ormore growth factors can also be added to this fluid to expose themicrografts 120 to such growth factors before implanting them in thematrix 210 or directly into the recipient site 220, which can enhancethe viability of the micrografts 120.

Force sensors, optical sensors, and/or position sensors can optionallybe provided in communication with the actuators 1120, 1130 and/or thetubes 510, 1010 and/or pin 1040, to improve control of the exemplaryharvesting and/or matrix implantation procedures described herein. Forexample, such sensor can be used to detect penetration depths and/orpenetration resistance of the tube 101 and/or the pin 1040 to assist inharvesting and/or implanting particular layers of tissue and/or sizes ofthe micrografts 120.

A sensor for detecting the presence of micrografts within the tube 510,1010 can also be provided. Such sensor can include, e.g., a source of asmall electric current provided to the tube 510, 1010 that is configuredto detect an electrical resistance or change in resistance within thetube 510, 1010. For example, a detected resistance when a small currentis flowed to the needle 510, 1010 (e.g., in an electrode configuration)can indicate whether the needle 510, 1010 is empty or if a micrograft120 is present therein. Alternatively, a laser fiber and a photodetectormay be provided within the needle to optically detect changes inscattered light, indicating whether or not the micrograft 120 ispresent.

Such micrograft sensor could be used, for example, to determine thenumber or percentage of actual micrografts harvested and/or implanted bythe tube 510, 1010, e.g., if a plurality of such micrografts isprocessed in a two-dimensional scan or traversal of the donor site 100and/or the recipient site 1510. A relatively small number of “missed”micrografts may be acceptable in a certain procedure, whereas a largernumber or percentage of “missed” micrografts can indicate, e.g., thatthe needle 510, 1010 needs to be replaced, that the graft material isnot viable or acceptable, and/or that the procedure should be repeatedor continued. If detected “missed” micrografts are localized in thedonor site 100, this could indicate that the tissue might bestructurally different in a particular region, e.g., a mole or smallscar can be present in the donor site 100.

An exemplary set of images of the micrograft that was collected from pigskin and placed in a collagen gel is shown in FIG. 16. The epidermalportion of the micrograft is the darker area at the lower end of themicrograft in these images. Even at times as short as 12 hours (secondimage from the left in FIG. 16), live cells can be observed migratingfrom the micrograft into the surrounding gel matrix, as indicated by thelighter areas around the dark micrograft. This migration is observed tocontinue 72 hours after placing the micrograft into the collagen gel(rightmost image in FIG. 16), indicating that the micrografts that areharvested and placed in a matrix, in accordance with exemplaryembodiments of the disclosure described herein, can be viable forextended times and can provide viable graft material.

In further exemplary embodiments of the disclosure, micrografts 120 canbe implanted into cleaned wound area 220 without the matrix. Forexample, FIG. 17 shows a series of exemplary images showing healing of awound generated in a nude (hairless) mouse, where micrografts obtainedfrom the skin of a black mouse were implanted in the wound area and thewound was then allowed to heal. After about 6 weeks, the wound appearsto be well-healed, and some tufts of black hair are observable. Theappearance of these tufts of black hair on the nude recipient sitesuggest that at least some of the micrografts survived during thehealing process and that functional hair follicles were successfullytransplanted to the nude recipient.

FIG. 18A shows an exemplary wound formed in a swine subject bysurgically removing a substantially square region of full-thickness skintissue (epidermis and dermis, down to the subcutaneous fat layer). Thesize of this wound is approximately 1.5 cm×1.5 cm. Micrografts wereharvested from a donor site of the swine subject using a device similarto the apparatus 500 shown in FIG. 5A, in accordance with the exemplaryembodiments described herein. The micrografts were implanted directlyinto one such wound (without using a matrix). A second similar wound wasalso generated in the subject and allowed to heal without implantationof any micrografts.

The upper row of FIG. 18B illustrates a series of exemplary imagesshowing the healing progress over 4 weeks for the wound shown in FIG.18A that had micrografts implanted therein. The lower row of FIG. 18Bare exemplary images showing the healing progress over 4 weeks for thewound that had no micrografts. The amount of observed wound contractionappears substantially reduced in the wound that had micrograftsimplanted in the wound site. In contrast, the wound without micrograftsappears to have contracted more significantly during the healingprocess. Contraction of skin tissue as a wound heals is generallyundesirable. For example, contraction of the skin around a joint canreduce the range of motion of the joint and may also be painful when thejoint is flexed or extended. In severe cases, the joint may be renderedsubstantially or completely immobile (e.g., severe tissue contractionaround the temporomandibular joints may prevent opening of a subject'smouth, and may require a liquid diet). Accordingly, the implantation ofmicrografts into a wound area as described herein may reduce tissuecontraction during wound healing and thereby reduce or avoid detrimentalside effects of such contraction.

The exemplary methods and apparatus described herein can also be used toharvest other types of biological tissue using the exemplary methods andapparatus described herein, and need not be limited to skin. Embodimentsof the present disclosure can facilitate harvesting of small tissueportions (e.g. micrografts 120) from various organs or tissue typeswhile reducing or avoiding generation of damage in the donor site. Theharvested tissue portions can provide viable tissue that may be used invarious grafting or cultivation procedures.

The foregoing merely illustrates the principles of the presentdisclosure. Various modifications and alterations to the describedembodiments will be apparent to those skilled in the art in view of theteachings herein. Various exemplary embodiments described herein can beused with one another interchangeably. It will thus be appreciated thatthose skilled in the art will be able to devise numerous techniqueswhich, although not explicitly described herein, embody the principlesof the present disclosure and are thus within the spirit and scope ofthe present disclosure. All patents and publications cited herein areincorporated herein by reference in their entireties.

What is claimed is:
 1. A system for obtaining a plurality of portions oftissue from a biological tissue, comprising: a plurality of tubescoupled to a base, each tube of the plurality comprising at least twopoints provided at a distal end thereof; a plurality of pins eachprovided at least partially within a central lumen of each of theplurality of tubes; a vibrating arrangement coupled to the baseconfigured to cause the plurality of tubes to vibrate and facilitate theinsertion of the tubes into the biological tissue, wherein at least onesection of the tubes is structured to be inserted into the biologicaltissue at the donor site to simultaneously remove the portions of tissuetherefrom when the at least one section of the tubes is withdrawn fromthe donor site, and wherein the pins are configured to facilitate aremoval of the portions of tissue from the tubes.
 2. The system of claim1, wherein an inner diameter of the tubes is less than about 1 mm. 3.The system of claim 1, wherein an inner diameter of the tubes is lessthan about 0.5 mm.
 4. The system of claim 1, wherein at least one of theplurality of tubes comprises at least three points provided at thedistal end thereof.
 5. The system of claim 1, further comprising atleast one positioning arrangement configured to control a position of atleast one of the plurality of pins within at least one of the pluralityof tubes.
 6. The system of claim 1, further comprising an actuatorconfigured to control a position of at least one of the plurality ofpins within at least one of the plurality of tubes.
 7. The system ofclaim 1, wherein at least one of the plurality of pins is controllablytranslatable in a direction along a longitudinal axis of the pluralityof tubes.
 8. The system of claim 1, wherein the plurality of tubescomprises at least six tubes.
 9. The system of claim 8, wherein thetubes are provided in at least one of a rectangular array or a linearconfiguration with respect to one another.
 10. The system of claim 1,further comprising at least one sensor arrangement configured to detecta presence of the portion of tissue within at least one of the pluralityof tubes.
 11. The system of claim 1, wherein an angle between a lateralside of the points and a longitudinal axis of at least one of theplurality of tubes is less than about 15 degrees.
 12. The system ofclaim 1, wherein the vibrating arrangement has an amplitude of vibrationof between 50 μm and 500 μm, and a frequency of vibrations between 10 Hzand 10 kHz.
 13. The system of claim 12, wherein the vibratingarrangement includes circuitry configured to adjust the amplitude andfrequency of the vibrations.
 14. The system of claim 1, wherein thesystem further comprises a biocompatible matrix configured to be placedon a donor site associated with the biological tissue and configured toreceive at least one portion of the portions of tissue from thebiological tissue.
 15. The system of claim 14, wherein the biocompatiblematrix comprises at least one of polyactic acid, collagen, low-meltingagarose, hyaluronic acid, hyaluranon, or devitalized skin tissue. 16.The system of claim 15, wherein the biocompatible matrix further anutrient, a growth factor, or a combination thereof.
 17. The system ofclaim 1, wherein at least a portion of a surface of the pins comprises alow-friction material.
 18. A method for obtaining at least one portionof at least one biological tissue, comprising: positioning a distal endof at least one hollow tube proximal to a donor tissue site; positioninga pin within a lumen of the at least one tube such that the distal endof the pin is proximal to the distal end of the at least one tube;advancing the at least one tube into the donor site to sever the atleast one portion of the at least one biological tissue from the donortissue site, wherein the at least one tube is coupled to a basecomprising a vibrating arrangement; actuating the vibrating arrangementto cause the at least one tube to vibrate to facilitate the insertion ofthe tubes into the biological tissue; and raising the at least one tubefrom the donor site, such that the at least one portion of the at leastone biological tissue is removed from the donor site.
 19. The method ofclaim 18, further comprising providing a matrix material over the donortissue site.
 20. A method for harvesting and implanting at least oneportion of at least one biological tissue, comprising: positioning adistal end of at least one hollow tube proximal to an upper surface ofthe at least one biological tissue; positioning a pin within a lumen ofthe at least one tube such that the distal end of the pin is provided ata predetermined distance behind the distal end of the at least one tube;advancing the at least one tube into the at least one biological tissueto sever the at least one portion of the at least one biological tissuefrom a surrounding tissue, such that the distal end of the pin ispositioned is proximal to the upper surface of the at least onebiological tissue, wherein the at least one tube is coupled to a base,actuating a vibrating arrangement coupled to the base to cause the atleast one tube to vibrate; raising the at least one tube and the pinsimultaneously until the at least one tube is removed from the at leastone biological tissue, wherein the at least one portion of the at leastone biological tissue remains within the at least one tube; inserting ahollow needle into a recipient material to a particular depth; providingthe at least one hollow tube containing the at least one portion of theat least one biological tissue in a lumen of the hollow needle, suchthat a distal end of the hollow tube is proximal to the distal end ofthe at least one hollow tube; retracting the hollow needle from therecipient material while maintaining a location of the at least onehollow tube within the recipient material; and withdrawing the at leastone hollow tube from the recipient material while maintaining a locationof the pin substantially stationary relative to the recipient material,such that the at least one portion of the at least one biological tissueremains within the recipient material.
 21. The method of claim 20,wherein the recipient material comprises at least one of a biologicallycompatible matrix or a further biological tissue.